Effects Of Osthole On Progesterone Secretion In Layer Chicken Preovulatory Follicles Granulosa Cells

Objective: Effects of osthole(Ost)on progesterone(P4)secretion was investigated in cultured granulosa cells obtained from hen preovulatory follicles.Methods: After separating the graded follicle granulosa cells from the 200-day-old Hyland brown hen,the cells were identified by HE staining and indirect immunofluorescence.The effect of Ost on granulosa cell viability was determined by MTT.After adding different concentrations(5,2.5,and 1.25 μg/m L)of Ost to granulosa cells for 6,12,18,and 24 h,the progesterone were detected by ELISA.After adding different concentrations(5,2.5,and 1.25 μg/m L)of Ost to granulosa cells for 24 h,the cyclic adenosine monophosphate(c AMP)and protein kinase A(PKA)were detected by ELISA;the gene expression of the steroidogenic acute regulatory protein(St AR),cytochrome P450 side-chain cleavage(P450scc),3-hydroxysteroid dehydrogenase(3β-HSD)and proliferating cell nuclear antigen(PCNA)were detected by q RT-PCR;the protein expression of St AR,P450 scc and 3β-HSD were detected by western blot;the level of reactive oxygen species(ROS)were detected by ROS kit.Results: 1.The isolated cells were identified as fusiform or polygonal by HE staining,and FSHR was highly expressed in cells identified by indirect immunofluorescence.2.The progesterone detected by ELISA was significantly increased in cells treated with 2.5 μg/m L of Ost for 24 h(P<0.05).3.q RT-PCR showed that gene expression of the St AR was significantly up-regulated by osthole at the concentration of 2.5 μg/m L(P<0.05);P450scc and 3β-HSD was significantly up-regulated by Ost(P<0.05).The protein expression of St AR,P450 scc and 3β-HSD by western blot was significantly up-regulated by Ost treatment(P<0.05).4.There was no significant effect on the expression of PCNA(P>0.05);ELISA showed that PKA was significantly up-regulated by Ost at 2.5 μg/m L(P<0.05),but not c AMP(P>0.05);ROS kit showed there was no significant difference in the level of ROS(P>0.05).Conclusions: 2.5 μg/m L Ost could significantly stimulate P4 secretion by up-regulation of PKA,P450 scc,3β-HSD and St AR genes in chicken preovulatory follicles granulosa cells after 24 h incubation.